An investigation of enzyme mechanisms using substrate analogues

Reference: Potter, Barry V. L., (1980). An investigation of enzyme mechanisms using substrate analogues. Dphil. University of Oxford.Citable link to this page:

An investigation of enzyme mechanisms using substrate analogues

Abstract: ﻿The synthesis of several fluorinated analogues of thiamine andprecursors has been undertaken, all of which exhibit weak activityagainst E.Coli. A route to 3-fluoro-(R)-alanine has been explored.The first synthesis of the enantiomeric fluorosuccinic acids hasbeen accomplished, and a stereochemical analysis has shown thatsynthesis via esterified malate precursors using diethylamino-sulphurtrifluoride gives inversion of configuration, whereas prep-aration via aspartic acid and polyhydrogen fluoride in pyridinegives retention of configuration. The circular dichroism spectraof the fluorosuccinic acids are anomalous, being the first exampleof such behaviour in a-substituted carboxylic acids and derivatives.A previously assigned configuration of a Pseudomonal metabolite ( + )-fluorosuccinic acid has been corrected. The fluorosuccinic acidsare not substrates for malic enzyme, but are potent inhibitors offumarase. Using (2S)-fluorosuccinic acid as a mechanistic probethe fumarase reaction is identified as a bimolecular E2 elimination.A stereochemical analysis of enzymic phosphoryl transferreactions using CD spectroscopy has been investigated, and a newsynthesis of inorganic pyrophosphate used to prepare P1=[(S)-160, 170, 180]-pyrophosphate whose 31P n.m.r. spectrum is discussed.The first example of 31 p- 17 o coupling as observed by 31 P n.m.r.is demonstrated.- The magnitude of the 31P- 180 isotope shift isdependent on the nature of the phosphorus to oxygen bond. Anelegant method for establishing the isotopic configuration of oxygenchiral phosphates by n.m.r. has been developed, requiring thesynthesis of D-glucose-6[(S) - 160, 170, 180]-phosphate, adenosine-5'-[(S)- 160, 170, 180]-phosphate, and their six membered cyclic phosphatetriesters. Chemical cyclisation of these molecules occurs bothwith racemisation and inversion of configuration at phosphorus.The chemical synthesis of adenosine-5'[ (S) - 160, 170, 180]-triphosphate has been achieved and exploited to demonstrate thathexokinase phosphoryl transfer proceeds with inversion of config-uration at phosphorus. Pyruvate kinase phosphoryl transfer from2 [(S)- 160, 170, 180]-phospho- (R)-glycerate also proceeds with inversionof configuration, as does phosphofructokinase phosphoryl transferfrom sn-glycerol-3[ (5)- 160, 170, 180]-phosphate.

Type of Award:Dphil Level of Award:Doctoral Awarding Institution: University of Oxford Notes:The digital copy of this thesis has been made available thanks to the generosity of Dr Leonard Polonsky